Assessment of Cell Viability in Intact Glandular Tissue in Chironomus Ramosus Using Dye-exclusion and Colorimetric Assays

Overview

Journal Cytotechnology

Specialties Biotechnology
Genetics

Date 2008 Nov 13

PMID 19003063

Authors

B B Nath

A A Babrekar

B Parthasarathy

Affiliations

Soon will be listed here.

Abstract

Conventionally, dye-exclusion test for determining cell viability has been restricted only for cells in suspension in tissue culture. In this paper, salivary gland of Chironomus has been proposed as a simple tissue model system where dye-exclusion test can be reliably employed for the intact gland. We have compared suitability of commonly used vital dyes and nigrosin was found suitable for the salivary gland cells. Biochemical tests using tetrazolium salts are also commonly used for determining quantitative indices of cell viability in metabolically active cells. Ours is the first attempt to extend the same technique for the whole tissue. We standardized the conditions and prepared a protocol for MTT-based colorimetric assay suitable for the salivary gland of Chironomus. A strong correlation (r(2) = 0.9893) was obtained where increasing O.D. correlated linearly with the number of live glands. We concluded that nigrosin dye-exclusion and MTT metabolic inclusion assays are suitable methods for the viability test of metabolically active intact salivary gland of Chironomus which can serve as a potential model for the assessment of cytotoxicity in future.

References

Krause A, Carley W, Webb W . Fluorescent erythrosin B is preferable to trypan blue as a vital exclusion dye for mammalian cells in monolayer culture. J Histochem Cytochem. 1984; 32(10):1084-90. DOI: 10.1177/32.10.6090533. View

Mosmann T . Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983; 65(1-2):55-63. DOI: 10.1016/0022-1759(83)90303-4. View

Bernas T, Dobrucki J . Mitochondrial and nonmitochondrial reduction of MTT: interaction of MTT with TMRE, JC-1, and NAO mitochondrial fluorescent probes. Cytometry. 2002; 47(4):236-42. DOI: 10.1002/cyto.10080. View

Bank H . Assessment of islet cell viability using fluorescent dyes. Diabetologia. 1987; 30(10):812-6. DOI: 10.1007/BF00275748. View

Olinga P, Groen K, Hof I, De Kanter R, Koster H, Leeman W . Comparison of five incubation systems for rat liver slices using functional and viability parameters. J Pharmacol Toxicol Methods. 1997; 38(2):59-69. DOI: 10.1016/s1056-8719(97)00060-9. View

KALTENBACH J, KALTENBACH M, Lyons W . Nigrosin as a dye for differentiating live and dead ascites cells. Exp Cell Res. 1958; 15(1):112-7. DOI: 10.1016/0014-4827(58)90067-3. View

Traore H, Meyer D . Comparing qualitative and quantitative spectroscopic techniques for the detection of the effect of direct iron loading of mammalian cell cultures. Methods Cell Sci. 2002; 23(4):175-84. DOI: 10.1023/a:1016313221027. View

Jones K, Senft J . An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide. J Histochem Cytochem. 1985; 33(1):77-9. DOI: 10.1177/33.1.2578146. View

Yamane H, Konishi K, Iguchi H, Nakagawa T, Shibata S, Takayama M . Assessment of hair cell death using the dye extrusion method. Acta Otolaryngol Suppl. 1999; 538:7-11. View

10.

Stevens M, Olsen S . Comparative analysis of using MTT and XTT in colorimetric assays for quantitating bovine neutrophil bactericidal activity. J Immunol Methods. 1993; 157(1-2):225-31. DOI: 10.1016/0022-1759(93)90091-k. View

11.

Doukas A, Flotte T . Physical characteristics and biological effects of laser-induced stress waves. Ultrasound Med Biol. 1996; 22(2):151-64. DOI: 10.1016/0301-5629(95)02026-8. View

12.

Tada H, Shiho O, Kuroshima K, Koyama M, Tsukamoto K . An improved colorimetric assay for interleukin 2. J Immunol Methods. 1986; 93(2):157-65. DOI: 10.1016/0022-1759(86)90183-3. View

13.

Drixler T, Vogten M, Ritchie E, van Vroonhoven T, Gebbink M, Voest E . Liver regeneration is an angiogenesis- associated phenomenon. Ann Surg. 2002; 236(6):703-11; discussion 711-2. PMC: 1422636. DOI: 10.1097/00000658-200212000-00002. View

14.

Grossbach U . The salivary gland of Chironomus (Diptera): a model system for the study of cell differentiation. Results Probl Cell Differ. 1977; 8:147-96. DOI: 10.1007/978-3-540-37332-2_3. View

15.

Means T, Geroski D, Hadley A, Lynn M, Edelhauser H . Viability of human corneal endothelium following Optisol-GS storage. Arch Ophthalmol. 1995; 113(6):805-9. DOI: 10.1001/archopht.1995.01100060131047. View

16.

Tan A, Berridge M . Superoxide produced by activated neutrophils efficiently reduces the tetrazolium salt, WST-1 to produce a soluble formazan: a simple colorimetric assay for measuring respiratory burst activation and for screening anti-inflammatory agents. J Immunol Methods. 2000; 238(1-2):59-68. DOI: 10.1016/s0022-1759(00)00156-3. View

17.

Da Costa A, de Assis M, Marques E, Plotkowski M . Comparative analysis of three methods to assess viability of mammalian cells in culture. Biocell. 2000; 23(1):65-72. View

18.

Loo D, Rillema J . Measurement of cell death. Methods Cell Biol. 1998; 57:251-64. DOI: 10.1016/s0091-679x(08)61583-6. View